The quality assurance of ELISA kits is a complex process, and several critical factors influence the accuracy and reliability of the test results.
First, the methodology plays a significant role. The ELISA assay can be performed using various techniques, such as the double-antibody sandwich method, indirect method, double-antigen sandwich method, IgM antibody capture method, and competitive inhibition method. Among these, the competitive inhibition method (used for HBeAb, HBcAb, etc.) may lead to less reproducible and qualitative results due to timing differences during the procedure. This method is more sensitive to variations in incubation times and other operational conditions, making it harder to control the outcome consistently.
Second, reagent quality is a crucial factor. Variations between different batches of ELISA reagents can affect performance, even if they pass batch testing. It’s essential to use long-batch reagents and ensure proper storage conditions. Following strict protocols helps avoid the need for re-establishing quality control systems or re-evaluating reagents when the batch number changes. For reagents that are used infrequently, it's advisable to dispense small amounts for each use to minimize repeated freeze-thaw cycles, which can degrade the reagent.
Safety precautions are also important during the experiment:
1. Avoid direct contact with stop solution and substrates A and B. If exposed, rinse immediately with water.
2. Do not eat, drink, smoke, or use cosmetics while working with the kit.
3. Never use your mouth to pipette any components of the kit.
Third, sample-related factors can significantly impact the results. Interference from endogenous substances—such as rheumatoid factor, complement, heterophilic antibodies, autoantibodies, and lysozyme—can cause false positives or negatives. Exogenous factors, like hemolysis, bacterial contamination, prolonged storage, incomplete clotting, or repeated freezing and thawing of samples, can also affect the accuracy of the test.
Coating is a key step in the ELISA process, where antigens or antibodies are immobilized onto a solid support, typically polystyrene plates. This binding occurs through physical adsorption, relying on hydrophobic interactions between the protein and the surface. Larger proteins tend to bind more effectively due to their higher number of hydrophobic groups. For example, IgG has a strong affinity for polystyrene, usually attaching via its Fc region, leaving the antigen-binding sites exposed. Most protein antigens can be coated similarly. However, non-protein antigens, such as DNA, may require special coating methods.
To enhance the binding of nucleic acids to polystyrene, the plate can be pre-treated with UV light (e.g., 30 W lamp for 75 hours at 75 cm) or coated with basic proteins like polylysine or protamine. Another approach is using the avidin-biotin system, where avidin is first coated onto the plate, followed by biotinylated antigens. This method ensures a uniform and stable coating, widely used for quantitative detection of various antigens.
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